Biosensors and Bioelectronics 71 (2015) 7–12
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
SERS-active Au@Ag nanorod dimers for ultrasensitive dopamine detection Lijuan Tang a, Si Li a, Fei Han b, Liqiang Liu a, Liguang Xu a, Wei Ma a, Hua Kuang a, Aike Li b,n, Libing Wang a, Chuanlai Xu a,n a b
State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, PR China Cereals & Oils Nutrition Research Group, Academy of Science & Technology of State Administration of Grain, Beijing 100037, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 5 February 2015 Received in revised form 24 March 2015 Accepted 5 April 2015 Available online 7 April 2015
Dopamine (DA) is a neurotransmitter which plays a key role in the life science. Self-assembled Au@Ag nanorod dimers based on aptamers were developed for ultrasensitive dopamine detection. The electronic field was significantly enhanced by the addition of silver shell coating on the surface of Au NR dimer. The results displayed that Au@Ag NR dimers were ideal building blocks for constructing the SERS substrates with prominent Raman enhancement effects. It was found that with using this Surface-enhanced Raman scattering (SERS)-encoded this sensing system, a limit of detection of 0.006 pM and a wide linear range of 0.01–10 pM for dopamine detection were obtained. Our work open up a new avenue for the diagnosis and drug-discovery programs. & 2015 Elsevier B.V. All rights reserved.
Keywords: Au@Ag nanorod dimers SERS Dopamine Detection
1. Introduction Dopamine (DA) is a neurotransmitter that regulates many physiological activities in the central nervous, hormonal, and cardiovascular systems (Lin et al., 2011). An abnormal level of DA in vivo can lead to serious illness, such as Alzheimer, Parkinson and Huntington diseases (Ahlskog, 2007; Tang et al., 2007) The concentration of DA in patients with these diseases is lower than that in healthy humans (Li et al., 2013). Various methods have been used for DA detection, including fluorescence (Yildirim and Bayindir, 2014), electrochemical (Farjami et al., 2013; Hao et al., 2012) and colorimetric biosensors (Lin et al., 2011), Surface-enhanced Raman scattering(SERS) (An et al., 2015; Balzerova et al., 2014; Bu and Lee, 2013; Qin, 2014), chromatography and others (Li et al., 2013; Park et al., 2014; Peterson et al., 2002; Zhang et al., 2013). Although these approaches are considerable advances in protein detection, they have several intrinsic drawbacks. For example, it is difficult to achieve high sensitivity for colorimetric biosensors. Chromatographic methods are expensive and timeconsuming, which restricts their wide clinical application. Moreover, electrochemical technology has some limitations related to specificity and stability due to interference from analogs, such as ascorbic acid, phenethylamine. Therefore, there is an urgent need n
Corresponding author. E-mail addresses: [email protected] (A. Li), [email protected] (C. Xu).
http://dx.doi.org/10.1016/j.bios.2015.04.013 0956-5663/& 2015 Elsevier B.V. All rights reserved.
to develop an ultrasensitive and highly selective analytical assay to monitor the level of DA in serum. Surface-enhanced Raman scattering (SERS), as a quantitative approach, is an extremely sensitive technique that can be tailored to provide the detection down to the single-molecule level (Ma et al., 2013b, 2014; Xu et al., 2012). SERS signal is enhanced by two main effects: electromagnetic and chemical enhancements (Graham et al., 1998). Some noble metallic materials, such as Au/Ag nanorods and Au/Ag nanoparticles, have been used as SERS substrates to generate high electromagnetic fields (Ma et al., 2013a; Shanthil et al., 2012; Zhu et al., 2012). However, the SERS signals derived from individual nanoparticles are too weak to apply in the ultrasensitive detection (Bell and Sirimuthu, 2008). To obtain high SERS signals, nanoparticle assemblies (e.g. dimers and trimers (Chen et al., 2010; Ma et al., 2013a, 2013b, 2014; Yan et al., 2012; Zhu et al., 2012)) with tunable gaps can improve electromagnetic fields and consequently generate the intense SERS signals. General, the smaller the gap, the stronger the signal is. Thus, in this study, we firstly used gold nanorods (Au NRs) to assemble NR dimers (Ma et al., 2013a; Xu et al., 2012), and then applied silver (Ag) shell coating on the surface of the dimers to achieve intense Raman signals (Wu et al., 2012). The Ag shell can not only reduce the gap between Au NRs, but also shows higher Raman enhancement than Au (Zhao et al., 2008). In addition, aptamers are single-strand RNA or DNA that have high affinity to targets, thermal stability, and reusability (Lu et al., 2010). Thus, aptamers, especially as the building blocks to assemble the nanocrystals as the probe (Cho et al., 2008; Wang
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L. Tang et al. / Biosensors and Bioelectronics 71 (2015) 7–12
2.3. Au nanorod (Au NR) synthesis Au NRs were synthesized by the seeds growth methodalal (Nikoobakht and El-Sayed, 2003). 2.3.1. Synthesis of Au seeds 2.5 mL, 0.5 mM hydrogen tetrachloroaurate (HAuCl4) was added to 2.5 mL, 0.2 M hexadecyltrimethylammonium bromide (CTAB) solution, then added 300 mL, 0.01 M sodium borohydride (NaBH4) and quickly mixed for 2 min and keep it at 25 °C. Scheme 1. SERS method for DA detection using Au@Ag NR dimers building by aptamer.
et al., 2010, 2007), have been widely used in detection techniques (Li et al., 2013; Ma et al., 2014, 2013c). In the present work, as shown in Scheme 1, DNA-1 and DNA-2 was coupled to the side of the Au NRs, respectively. The aptamer with specific affinity with DA was used, which can partially complement with single strand DNA (DNA-1, DNA-2) (Li et al., 2013). With the addition of aptamer, the Au NR dimer probe was formed following DNA hybridization. If DA molecules are present in solution, the high specific recognition of aptamer and DA will cause the dimer to dehybridize to form single Au NRs. The Raman intensity is in proportion to the yield of Au NR dimers in the sensing system. Therefore, the higher the DA concentration, the weaker the Raman intensity will be. To further enhance Raman signals, the colloid NRs were coated with an Ag shell and used as the SERS substrate. This could be significantly improved the sensitivity of detection.
2. Experimental section 2.1. Material Thiolated DNA aptamer was purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. The aptamer was purified by high-performance liquid chromatography (HPLC) and suspended in deionized water from a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France). Dopamine hydrochloride, catechol, phenethylamine were purchased from Aladdin. All other chemicals used in this work were obtained from Sigma-Aldrich. The detailed sequences of the oligonucleotides are as follows: DA-aptamer (aptamer): 5′-GTC TCT GTG TGC GCC AGA GAC ACT GGG GCA GAT ATG GGC CAG CAC AGA ATG AGG CCC-3′ DA-complementary-1 (DNA1): 3′-SH-CAG AGA CAC ACG-5′ DA-complementary-2 (DNA2): 3′-TCT TAC TCC GGG-SH-5′
2.3.2. The growth of Au NR 5 mL, 1 mM HAuCl4 was added to 5 mL of 0.2 M CTAB solution, then slowly added 0.15 mL of 4 mM AgNO3 and left to reaction for 5 min, then added 70 mL of 0.079 M ascorbic acid (Vc) and left it to reduce for 2 min, finally 12 mL of prepared Au seeds were added, strongly stirred for 20 s and left it at 25 °C for 2 h. The colloidal solution was concentrated by centrifugation at 7000 rpm for 30 min. The supernatants were removed and the precipitate was resuspended into 5 mM CTAB solution. The concentration of Au NRs was 10 nM according to the results from the inductively coupled plasma mass spectrometry. 2.4. Au NRs modification In order to obtain side-by-side oriented Au NR dimers, thiolated PEG1000 was used to modify onto the end facets of Au NRs at a PEG/NR molar ratio of 20:1 (Ma et al., 2013a). 50 mL aliquot of concentrated Au NRs was mixed with 50 mL, 10 mM Tris buffer, then added 2 mL of 5 mM PEG under vigorous stirring. After incubation for 10 h, the excess PEG was removed by centrifugation at 7000 rpm for 5 min and dissolved in 100 mL, 5 mM CTAB-10 mM Tris buffer. Consequently, the PEG-modified Au NRs was modified by the addition of 2 mL, 5 mM complementary DNA1 or DAN2. After incubation for 12 h, the excess DNA was removed by centrifugation (3 times) at 7000 rpm for 5 min. And, the Au NR was resuspended in 100 mL of 5 mM CTAB-10 mM Tris buffer and characterized by dynamic light scattering (DLS). The hydrodynamic size of the DNAmodified Au NRs increased, which demonstrated that DNA was successfully conjugated to the surface of the Au NRs and could be used for the subsequent assembly (Fig. S1). 2.5. Fabrication of side-by-side oriented Au NR dimers 50 mL of AuNR-DNA1 and 50 mL of AuNR-DNA2 was mixed with a ratio of 1:1, then added 2 mL of 2 mM aptamer and incubation for 8 h. The NR pairs were formed by adding the aptamers. Then, 4-ATP ethanol solution, with a final concentration of 10 mM was added to the mixtures and incubated at room temperature for more than 5 h. 2.6. Dopamine detection
2.2. Instrumentation Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-2100 operating at an acceleration voltage of 200 kV. All ultraviolet–visible (UV–vis) spectra were acquired using UNICO 2100 PC UV–vis spectrophotometer and processed with Origin Lab software. The dynamic light scattering (DLS) data were measured by Zetasizer Nano ZS system (Malvern) with 632.8 nm laser. Raman spectra were measured by LabRam-HR800 Micro-Raman spectrometer with Lab-spec 5.0 software attached to a liquid cell. The slit and pinhole were set at 100 and 400 mm, respectively, an aircooled He–Ne laser for 633 nm excitation with a power of 8 mW.
50 mL of AuNR-DNA1 (0.025 nM) and 50 mL of AuNR-DNA2 (0.025 nM) was mixed with aptamer (2 mL, 1 nM) in CTAB-Tris buffer (10 mM Tris, PH 7.4, 5 mM CTAB). After gentle shaking for several minutes, 1 mL of dopamine with various concentrations were respectively added to the solutions, resulting in final concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 pM. The solutions were then incubated for 8 h at 37 °C. Then the reacted mixture was coated with Ag shell. 1 μL of 4-ATP with final concentration of 10 μM was added. 2.7. Au@Ag NR dimer synthesis 100 mL of Au NR dimers was centrifuged at 7000 rpm for 5 min,
L. Tang et al. / Biosensors and Bioelectronics 71 (2015) 7–12
the precipitate were resuspended into 200 mL of 20 mM CTAB solution. Then, 20 mL (10–0.1 mM) AgNO3 and 10 mL of 0.1 M Vc were mixed with the solution. Finally, 20 mL of 10 mM NaOH was added to adjust the reacted solution to keep at pH 9. After reacting for 5 min, the mixtures were centrifuged at 6000 rpm for 5 min. The precipitate was then dissolved in 200 mL of deionized water. The thickness of Ag shell was adjusted by addition varying concentration of Ag NO3.
2.8. Electric field simulation of NR oriented side-by-side pairs Finite integration technique (FIT) simulation was used to calculate the electric field enhancement effect of NR oriented sideby-side before and after coating silver shell. An FIT package from CST STUDIO SUITE 2010 was explored to calculate the vicinity of the plasmonic for media with electrical permittivity of ε ¼1 and magnetic permeability of μe ¼1. NR dimers in the presence of 5 mM AgNO3 were modeled by 43 11 nm2 gold nanorod and 8.5 nm silver shell. The corresponding interparticle distance of NR dimers before and after silver shell coating was estimated to be 19 nm and 5 nm, respectively, which was consistent with the data measured by TEM. The local electromagnetic fields were calculated for 632.8 nm excitation and parallel orientation of rods to the external field.
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3. Results and discussion 3.1. Fabrication of high SERS-active Au@Ag dimers Initially, the Au NRs with length/diameter of 43 nm/11 nm, were used to assemble dimers. Firstly, the end surface of Au NRs were blocked by thiol modified polyethylene glycol 1000 (PEG 1000) at a PEG/NR molar ratio of 20:1. And then, DNA1 and DNA2 were coupled with the side of NR at a DNA/NR molar ratio of 20:1. The size of Au NRs before and after DNA conjunction changed which demonstrated that DNA was successfully modified onto the surface of the Au NRs (Fig. S1). According to the fluorescence methods (Hurst et al., 2006), the number of DNA1 and DNA2 coupled on NRs was calculated to be 1.270.3 and 1.1 7 0.1, respectively. Subsequently, formation of NR dimers was obtained by hybridization of complementary DNA strands at a NR-DNA1/NRDNA2/aptamer molar ratio of 1:1:1.6. To obtain the highest SERS signal, we compared the SERS intensity of Au@Ag NR dimers with different shell thickness synthesized by adding different concentrations of AgNO3. Transmission electron microscopy (TEM) showed that the Ag shell was thicker when the concentration of AgNO3 was increased. In addition, the gap distance between Au NRs in the dimers became shorter. The gap distance was more than 19 nm when the dimers were bare. However, when 10 mM AgNO3 was added, there was almost no gap between the dimers (Fig. 1). The color also changed markedly with Ag shell thickness, from gray to light green, green, purplish red, red and orange with increased Ag shell thickness (Fig. 2A). This result is consistent with previous study (Liu and
Fig. 1. TEM images of Au@Ag NR dimers with different Ag shell thickness by adding various concentrations of AgNO3: (A)0, (B)0.6 mM, (C)1.2 mM, (D)2.5 mM, (E)5 mM and (F) 10 mM.
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Fig. 2. Optical property of Au@Ag NR dimers with different Ag shell thickness by addition different concentration of AgNO3 in the range of 0–10 mM: (A) photographs; (B) ultraviolet–visible (UV–vis) spectra; (C) SERS spectra of 4-ATP anchored onto the surface of nanorods at 1075 cm 1; (D) the growth trend of intensity at 1075 cm 1.
Guyot-Sionnest, 2004). Moreover, the Ag shell had a marked effect on the local surface plasmon resonance (LSPR) of the Au NRs (Fig. 2B). As the Ag shell became thicker accompany with the concentration of AgNO3 increasing from 0 to 5 mM, the longitudinal LSPR gradually blue-shifted from 750 nm to 520 nm, however, the transverse LSPR red-shifted slightly. And, it should be noted that the LSPR of Ag also became stronger and red-shifted as the Ag shell became thicker. When the thickness increased above a certain value, all LSPR of the Au@Ag NR dimers merged together. To evaluate plasmon enhanced Raman effect of Au@Ag NR dimers with various thickness, a standard Raman label, 4-aminothiophenol (4-ATP), was attached to the silver shell coated Au NR dimers. The SERS spectra showed that the Raman intensity initially increased, then decreased with increased Ag shell thickness (Fig. 2C and D). The Raman intensity was maximum when 2.5 mM AgNO3 was added. At this Ag shell thickness (6.89 nm Ag shell thickness), the signal was approximately 6 times higher than that for bare dimers. Note that in spite of the intensity of Au@Ag NRs were also higher than the bare Au NRs, the signal was approximately 10 times weaker than the Au@Ag NR dimers (Fig. S2). Thus, we chose 2.5 mM AgNO3 to synthesize the silver shell of the NR SERS probe to detect DA. Moreover, E-field distributions were simulated for Au NR dimers and Au@Ag NR dimers, and the E-field intensity was 2.44 and 26.1, respectively, which further demonstrated that the Ag shell coating on the surface of the dimers can significantly enhance the electromagnetic field (Fig. S5).
3.2. Application of the as-fabricated sensor for DA detection As DA and NR–DNA competing to bind with aptamer, the concentration of DA in solution directly influenced the degree of self-assembly of Au NR dimers (Fig. S3). In this study, the characteristic SERS peak of 4-ATP at 1075 cm 1 was employed to quantify the concentration of DA. Fig. 3A shows that the solution without DA had the highest assembly yield, and with increased DA concentration, the dimers gradually decreased, and the Raman signal of the sensing systems decreased by degrees. A standard curve for DA detection was then plotted, which was based on the intensity of the 1075 cm 1 peak of 4-ATP against the logarithm of the DA concentration over the range of 0.01–10 pM (Fig. 3B), and displayed a superb correlation (R2 ¼0.9904). The fitted linear equation is y¼ 741.67–1316.83lg x. The limit of detection (LOD) was determined to be 0.006 pM, which is the lowest value reported to date (Table S1). The LOD of DA is determined based on a linear fitting and the noise level of 3s where s is the standard deviation of SERS intensity at 1075 cm 1 measured at 0 aM DA. Interestingly, as shown in Fig. S4, the UV–vis spectrum of the Au@Ag NR dimer sensor in different DA samples displayed slightly changed. 3.3. Selectivity of the sensor for DA detection To verify the selectivity of this assay, eight interfering chemicals (AA, CT, GL, Cys, TR and PEA, HSA, BSA) were tested under the same conditions (Li et al., 2013; Yildirim and Bayindir, 2014; Zhang
L. Tang et al. / Biosensors and Bioelectronics 71 (2015) 7–12
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et al., 2013). The DA concentration was 1 nM, the BSA and HSA concentration was 1 mM, and the concentration of the other substances was 1 μM. The SERS intensity of the Au@Ag NR dimer sensor containing 1 nM DA was approximately 5-fold lower than the control (without any target and interferences addition) (Fig. 4). More importantly, the SERS intensity showed no obvious reduction with these interfering substances even at this high concentration. To further confirm the specificity of this method, the all interfering chemicals, which were used in this assay, were mixed as the substrate for DA detection (Yildirim and Bayindir, 2014). The results revealed that the interfering chemicals had no influence on DA detection. Therefore, this method had excellent selectivity for the detection of DA.
4. Conclusions In summary, an ultrasensitive and highly selective method for the DA detection, based on aptamer-mediated dimer and coated with an Ag shell on the surface to further enhance the SERS signal, was developed for the first time. It was found that the Raman intensity initially increased, and then decreased with increased Ag shell thickness. The mechanism was revealed that the spatial configuration of rod dimers could be changed to form the large dihedral angle to reduce the surface plasmonic resonance coupling effects and further decreased the intensity of SERS signal as increasing the thickness of silver shells to reduce the electric repulsive of adjacent NRs. The LOD of 0.006 pM and broad linear range were obtained. We believe that this simple, low cost, ultrasensitive and high selective method could be used as a promising technology for the detection of DA deficiency-related diseases in the future.
Acknowledgments
Fig. 3. The SERS method for detection of DA. (A) The SERS spectra of Au@Ag NR dimers assemblies with different concentration of DA. (B) The standard curve for the DA detection, where the SERS intensities were calculated at the frequency of 1075 cm 1.
This work is financially supported by the National Natural Science Foundation of China (21471068, 31400848), the Key Programs from MOST (2012YQ09019410, 2012BAC01B07), and grants from Natural Science Foundation of Jiangsu Province (BE2013613, BE2013611). This research was also supported by the Special Fund for Grain Research in the Public Interest (201313011-6).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.013
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Fig. 4. Selectivity of this developed dopamine assay, DA concentration is 1 nM; BSA, HSA concentration is 1 mM; other substances are 1 μM (AA, ascorbic acid; CT, catechol; GL, glucose; Cys, L-cysteine; TR, tyrosine; PEA, phenethylamine; HSA, human serum albumin; BSA, bovine serum albumin; DA, dopamine; Mix, mixture of all interfering chemicals).
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
SERS-active Au@Ag nanorod dimers for ultrasensitive dopamine detection Lijuan Tang a, Si Li a, Fei Han b, Liqiang Liu a, Liguang Xu a, Wei Ma a, Hua Kuang a, Aike Li b,n, Libing Wang a, Chuanlai Xu a,n a b
State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, PR China Cereals & Oils Nutrition Research Group, Academy of Science & Technology of State Administration of Grain, Beijing 100037, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 5 February 2015 Received in revised form 24 March 2015 Accepted 5 April 2015 Available online 7 April 2015
Dopamine (DA) is a neurotransmitter which plays a key role in the life science. Self-assembled Au@Ag nanorod dimers based on aptamers were developed for ultrasensitive dopamine detection. The electronic field was significantly enhanced by the addition of silver shell coating on the surface of Au NR dimer. The results displayed that Au@Ag NR dimers were ideal building blocks for constructing the SERS substrates with prominent Raman enhancement effects. It was found that with using this Surface-enhanced Raman scattering (SERS)-encoded this sensing system, a limit of detection of 0.006 pM and a wide linear range of 0.01–10 pM for dopamine detection were obtained. Our work open up a new avenue for the diagnosis and drug-discovery programs. & 2015 Elsevier B.V. All rights reserved.
Keywords: Au@Ag nanorod dimers SERS Dopamine Detection
1. Introduction Dopamine (DA) is a neurotransmitter that regulates many physiological activities in the central nervous, hormonal, and cardiovascular systems (Lin et al., 2011). An abnormal level of DA in vivo can lead to serious illness, such as Alzheimer, Parkinson and Huntington diseases (Ahlskog, 2007; Tang et al., 2007) The concentration of DA in patients with these diseases is lower than that in healthy humans (Li et al., 2013). Various methods have been used for DA detection, including fluorescence (Yildirim and Bayindir, 2014), electrochemical (Farjami et al., 2013; Hao et al., 2012) and colorimetric biosensors (Lin et al., 2011), Surface-enhanced Raman scattering(SERS) (An et al., 2015; Balzerova et al., 2014; Bu and Lee, 2013; Qin, 2014), chromatography and others (Li et al., 2013; Park et al., 2014; Peterson et al., 2002; Zhang et al., 2013). Although these approaches are considerable advances in protein detection, they have several intrinsic drawbacks. For example, it is difficult to achieve high sensitivity for colorimetric biosensors. Chromatographic methods are expensive and timeconsuming, which restricts their wide clinical application. Moreover, electrochemical technology has some limitations related to specificity and stability due to interference from analogs, such as ascorbic acid, phenethylamine. Therefore, there is an urgent need n
Corresponding author. E-mail addresses: [email protected] (A. Li), [email protected] (C. Xu).
http://dx.doi.org/10.1016/j.bios.2015.04.013 0956-5663/& 2015 Elsevier B.V. All rights reserved.
to develop an ultrasensitive and highly selective analytical assay to monitor the level of DA in serum. Surface-enhanced Raman scattering (SERS), as a quantitative approach, is an extremely sensitive technique that can be tailored to provide the detection down to the single-molecule level (Ma et al., 2013b, 2014; Xu et al., 2012). SERS signal is enhanced by two main effects: electromagnetic and chemical enhancements (Graham et al., 1998). Some noble metallic materials, such as Au/Ag nanorods and Au/Ag nanoparticles, have been used as SERS substrates to generate high electromagnetic fields (Ma et al., 2013a; Shanthil et al., 2012; Zhu et al., 2012). However, the SERS signals derived from individual nanoparticles are too weak to apply in the ultrasensitive detection (Bell and Sirimuthu, 2008). To obtain high SERS signals, nanoparticle assemblies (e.g. dimers and trimers (Chen et al., 2010; Ma et al., 2013a, 2013b, 2014; Yan et al., 2012; Zhu et al., 2012)) with tunable gaps can improve electromagnetic fields and consequently generate the intense SERS signals. General, the smaller the gap, the stronger the signal is. Thus, in this study, we firstly used gold nanorods (Au NRs) to assemble NR dimers (Ma et al., 2013a; Xu et al., 2012), and then applied silver (Ag) shell coating on the surface of the dimers to achieve intense Raman signals (Wu et al., 2012). The Ag shell can not only reduce the gap between Au NRs, but also shows higher Raman enhancement than Au (Zhao et al., 2008). In addition, aptamers are single-strand RNA or DNA that have high affinity to targets, thermal stability, and reusability (Lu et al., 2010). Thus, aptamers, especially as the building blocks to assemble the nanocrystals as the probe (Cho et al., 2008; Wang
8
L. Tang et al. / Biosensors and Bioelectronics 71 (2015) 7–12
2.3. Au nanorod (Au NR) synthesis Au NRs were synthesized by the seeds growth methodalal (Nikoobakht and El-Sayed, 2003). 2.3.1. Synthesis of Au seeds 2.5 mL, 0.5 mM hydrogen tetrachloroaurate (HAuCl4) was added to 2.5 mL, 0.2 M hexadecyltrimethylammonium bromide (CTAB) solution, then added 300 mL, 0.01 M sodium borohydride (NaBH4) and quickly mixed for 2 min and keep it at 25 °C. Scheme 1. SERS method for DA detection using Au@Ag NR dimers building by aptamer.
et al., 2010, 2007), have been widely used in detection techniques (Li et al., 2013; Ma et al., 2014, 2013c). In the present work, as shown in Scheme 1, DNA-1 and DNA-2 was coupled to the side of the Au NRs, respectively. The aptamer with specific affinity with DA was used, which can partially complement with single strand DNA (DNA-1, DNA-2) (Li et al., 2013). With the addition of aptamer, the Au NR dimer probe was formed following DNA hybridization. If DA molecules are present in solution, the high specific recognition of aptamer and DA will cause the dimer to dehybridize to form single Au NRs. The Raman intensity is in proportion to the yield of Au NR dimers in the sensing system. Therefore, the higher the DA concentration, the weaker the Raman intensity will be. To further enhance Raman signals, the colloid NRs were coated with an Ag shell and used as the SERS substrate. This could be significantly improved the sensitivity of detection.
2. Experimental section 2.1. Material Thiolated DNA aptamer was purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. The aptamer was purified by high-performance liquid chromatography (HPLC) and suspended in deionized water from a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France). Dopamine hydrochloride, catechol, phenethylamine were purchased from Aladdin. All other chemicals used in this work were obtained from Sigma-Aldrich. The detailed sequences of the oligonucleotides are as follows: DA-aptamer (aptamer): 5′-GTC TCT GTG TGC GCC AGA GAC ACT GGG GCA GAT ATG GGC CAG CAC AGA ATG AGG CCC-3′ DA-complementary-1 (DNA1): 3′-SH-CAG AGA CAC ACG-5′ DA-complementary-2 (DNA2): 3′-TCT TAC TCC GGG-SH-5′
2.3.2. The growth of Au NR 5 mL, 1 mM HAuCl4 was added to 5 mL of 0.2 M CTAB solution, then slowly added 0.15 mL of 4 mM AgNO3 and left to reaction for 5 min, then added 70 mL of 0.079 M ascorbic acid (Vc) and left it to reduce for 2 min, finally 12 mL of prepared Au seeds were added, strongly stirred for 20 s and left it at 25 °C for 2 h. The colloidal solution was concentrated by centrifugation at 7000 rpm for 30 min. The supernatants were removed and the precipitate was resuspended into 5 mM CTAB solution. The concentration of Au NRs was 10 nM according to the results from the inductively coupled plasma mass spectrometry. 2.4. Au NRs modification In order to obtain side-by-side oriented Au NR dimers, thiolated PEG1000 was used to modify onto the end facets of Au NRs at a PEG/NR molar ratio of 20:1 (Ma et al., 2013a). 50 mL aliquot of concentrated Au NRs was mixed with 50 mL, 10 mM Tris buffer, then added 2 mL of 5 mM PEG under vigorous stirring. After incubation for 10 h, the excess PEG was removed by centrifugation at 7000 rpm for 5 min and dissolved in 100 mL, 5 mM CTAB-10 mM Tris buffer. Consequently, the PEG-modified Au NRs was modified by the addition of 2 mL, 5 mM complementary DNA1 or DAN2. After incubation for 12 h, the excess DNA was removed by centrifugation (3 times) at 7000 rpm for 5 min. And, the Au NR was resuspended in 100 mL of 5 mM CTAB-10 mM Tris buffer and characterized by dynamic light scattering (DLS). The hydrodynamic size of the DNAmodified Au NRs increased, which demonstrated that DNA was successfully conjugated to the surface of the Au NRs and could be used for the subsequent assembly (Fig. S1). 2.5. Fabrication of side-by-side oriented Au NR dimers 50 mL of AuNR-DNA1 and 50 mL of AuNR-DNA2 was mixed with a ratio of 1:1, then added 2 mL of 2 mM aptamer and incubation for 8 h. The NR pairs were formed by adding the aptamers. Then, 4-ATP ethanol solution, with a final concentration of 10 mM was added to the mixtures and incubated at room temperature for more than 5 h. 2.6. Dopamine detection
2.2. Instrumentation Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-2100 operating at an acceleration voltage of 200 kV. All ultraviolet–visible (UV–vis) spectra were acquired using UNICO 2100 PC UV–vis spectrophotometer and processed with Origin Lab software. The dynamic light scattering (DLS) data were measured by Zetasizer Nano ZS system (Malvern) with 632.8 nm laser. Raman spectra were measured by LabRam-HR800 Micro-Raman spectrometer with Lab-spec 5.0 software attached to a liquid cell. The slit and pinhole were set at 100 and 400 mm, respectively, an aircooled He–Ne laser for 633 nm excitation with a power of 8 mW.
50 mL of AuNR-DNA1 (0.025 nM) and 50 mL of AuNR-DNA2 (0.025 nM) was mixed with aptamer (2 mL, 1 nM) in CTAB-Tris buffer (10 mM Tris, PH 7.4, 5 mM CTAB). After gentle shaking for several minutes, 1 mL of dopamine with various concentrations were respectively added to the solutions, resulting in final concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 pM. The solutions were then incubated for 8 h at 37 °C. Then the reacted mixture was coated with Ag shell. 1 μL of 4-ATP with final concentration of 10 μM was added. 2.7. Au@Ag NR dimer synthesis 100 mL of Au NR dimers was centrifuged at 7000 rpm for 5 min,
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the precipitate were resuspended into 200 mL of 20 mM CTAB solution. Then, 20 mL (10–0.1 mM) AgNO3 and 10 mL of 0.1 M Vc were mixed with the solution. Finally, 20 mL of 10 mM NaOH was added to adjust the reacted solution to keep at pH 9. After reacting for 5 min, the mixtures were centrifuged at 6000 rpm for 5 min. The precipitate was then dissolved in 200 mL of deionized water. The thickness of Ag shell was adjusted by addition varying concentration of Ag NO3.
2.8. Electric field simulation of NR oriented side-by-side pairs Finite integration technique (FIT) simulation was used to calculate the electric field enhancement effect of NR oriented sideby-side before and after coating silver shell. An FIT package from CST STUDIO SUITE 2010 was explored to calculate the vicinity of the plasmonic for media with electrical permittivity of ε ¼1 and magnetic permeability of μe ¼1. NR dimers in the presence of 5 mM AgNO3 were modeled by 43 11 nm2 gold nanorod and 8.5 nm silver shell. The corresponding interparticle distance of NR dimers before and after silver shell coating was estimated to be 19 nm and 5 nm, respectively, which was consistent with the data measured by TEM. The local electromagnetic fields were calculated for 632.8 nm excitation and parallel orientation of rods to the external field.
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3. Results and discussion 3.1. Fabrication of high SERS-active Au@Ag dimers Initially, the Au NRs with length/diameter of 43 nm/11 nm, were used to assemble dimers. Firstly, the end surface of Au NRs were blocked by thiol modified polyethylene glycol 1000 (PEG 1000) at a PEG/NR molar ratio of 20:1. And then, DNA1 and DNA2 were coupled with the side of NR at a DNA/NR molar ratio of 20:1. The size of Au NRs before and after DNA conjunction changed which demonstrated that DNA was successfully modified onto the surface of the Au NRs (Fig. S1). According to the fluorescence methods (Hurst et al., 2006), the number of DNA1 and DNA2 coupled on NRs was calculated to be 1.270.3 and 1.1 7 0.1, respectively. Subsequently, formation of NR dimers was obtained by hybridization of complementary DNA strands at a NR-DNA1/NRDNA2/aptamer molar ratio of 1:1:1.6. To obtain the highest SERS signal, we compared the SERS intensity of Au@Ag NR dimers with different shell thickness synthesized by adding different concentrations of AgNO3. Transmission electron microscopy (TEM) showed that the Ag shell was thicker when the concentration of AgNO3 was increased. In addition, the gap distance between Au NRs in the dimers became shorter. The gap distance was more than 19 nm when the dimers were bare. However, when 10 mM AgNO3 was added, there was almost no gap between the dimers (Fig. 1). The color also changed markedly with Ag shell thickness, from gray to light green, green, purplish red, red and orange with increased Ag shell thickness (Fig. 2A). This result is consistent with previous study (Liu and
Fig. 1. TEM images of Au@Ag NR dimers with different Ag shell thickness by adding various concentrations of AgNO3: (A)0, (B)0.6 mM, (C)1.2 mM, (D)2.5 mM, (E)5 mM and (F) 10 mM.
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Fig. 2. Optical property of Au@Ag NR dimers with different Ag shell thickness by addition different concentration of AgNO3 in the range of 0–10 mM: (A) photographs; (B) ultraviolet–visible (UV–vis) spectra; (C) SERS spectra of 4-ATP anchored onto the surface of nanorods at 1075 cm 1; (D) the growth trend of intensity at 1075 cm 1.
Guyot-Sionnest, 2004). Moreover, the Ag shell had a marked effect on the local surface plasmon resonance (LSPR) of the Au NRs (Fig. 2B). As the Ag shell became thicker accompany with the concentration of AgNO3 increasing from 0 to 5 mM, the longitudinal LSPR gradually blue-shifted from 750 nm to 520 nm, however, the transverse LSPR red-shifted slightly. And, it should be noted that the LSPR of Ag also became stronger and red-shifted as the Ag shell became thicker. When the thickness increased above a certain value, all LSPR of the Au@Ag NR dimers merged together. To evaluate plasmon enhanced Raman effect of Au@Ag NR dimers with various thickness, a standard Raman label, 4-aminothiophenol (4-ATP), was attached to the silver shell coated Au NR dimers. The SERS spectra showed that the Raman intensity initially increased, then decreased with increased Ag shell thickness (Fig. 2C and D). The Raman intensity was maximum when 2.5 mM AgNO3 was added. At this Ag shell thickness (6.89 nm Ag shell thickness), the signal was approximately 6 times higher than that for bare dimers. Note that in spite of the intensity of Au@Ag NRs were also higher than the bare Au NRs, the signal was approximately 10 times weaker than the Au@Ag NR dimers (Fig. S2). Thus, we chose 2.5 mM AgNO3 to synthesize the silver shell of the NR SERS probe to detect DA. Moreover, E-field distributions were simulated for Au NR dimers and Au@Ag NR dimers, and the E-field intensity was 2.44 and 26.1, respectively, which further demonstrated that the Ag shell coating on the surface of the dimers can significantly enhance the electromagnetic field (Fig. S5).
3.2. Application of the as-fabricated sensor for DA detection As DA and NR–DNA competing to bind with aptamer, the concentration of DA in solution directly influenced the degree of self-assembly of Au NR dimers (Fig. S3). In this study, the characteristic SERS peak of 4-ATP at 1075 cm 1 was employed to quantify the concentration of DA. Fig. 3A shows that the solution without DA had the highest assembly yield, and with increased DA concentration, the dimers gradually decreased, and the Raman signal of the sensing systems decreased by degrees. A standard curve for DA detection was then plotted, which was based on the intensity of the 1075 cm 1 peak of 4-ATP against the logarithm of the DA concentration over the range of 0.01–10 pM (Fig. 3B), and displayed a superb correlation (R2 ¼0.9904). The fitted linear equation is y¼ 741.67–1316.83lg x. The limit of detection (LOD) was determined to be 0.006 pM, which is the lowest value reported to date (Table S1). The LOD of DA is determined based on a linear fitting and the noise level of 3s where s is the standard deviation of SERS intensity at 1075 cm 1 measured at 0 aM DA. Interestingly, as shown in Fig. S4, the UV–vis spectrum of the Au@Ag NR dimer sensor in different DA samples displayed slightly changed. 3.3. Selectivity of the sensor for DA detection To verify the selectivity of this assay, eight interfering chemicals (AA, CT, GL, Cys, TR and PEA, HSA, BSA) were tested under the same conditions (Li et al., 2013; Yildirim and Bayindir, 2014; Zhang
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et al., 2013). The DA concentration was 1 nM, the BSA and HSA concentration was 1 mM, and the concentration of the other substances was 1 μM. The SERS intensity of the Au@Ag NR dimer sensor containing 1 nM DA was approximately 5-fold lower than the control (without any target and interferences addition) (Fig. 4). More importantly, the SERS intensity showed no obvious reduction with these interfering substances even at this high concentration. To further confirm the specificity of this method, the all interfering chemicals, which were used in this assay, were mixed as the substrate for DA detection (Yildirim and Bayindir, 2014). The results revealed that the interfering chemicals had no influence on DA detection. Therefore, this method had excellent selectivity for the detection of DA.
4. Conclusions In summary, an ultrasensitive and highly selective method for the DA detection, based on aptamer-mediated dimer and coated with an Ag shell on the surface to further enhance the SERS signal, was developed for the first time. It was found that the Raman intensity initially increased, and then decreased with increased Ag shell thickness. The mechanism was revealed that the spatial configuration of rod dimers could be changed to form the large dihedral angle to reduce the surface plasmonic resonance coupling effects and further decreased the intensity of SERS signal as increasing the thickness of silver shells to reduce the electric repulsive of adjacent NRs. The LOD of 0.006 pM and broad linear range were obtained. We believe that this simple, low cost, ultrasensitive and high selective method could be used as a promising technology for the detection of DA deficiency-related diseases in the future.
Acknowledgments
Fig. 3. The SERS method for detection of DA. (A) The SERS spectra of Au@Ag NR dimers assemblies with different concentration of DA. (B) The standard curve for the DA detection, where the SERS intensities were calculated at the frequency of 1075 cm 1.
This work is financially supported by the National Natural Science Foundation of China (21471068, 31400848), the Key Programs from MOST (2012YQ09019410, 2012BAC01B07), and grants from Natural Science Foundation of Jiangsu Province (BE2013613, BE2013611). This research was also supported by the Special Fund for Grain Research in the Public Interest (201313011-6).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.013
References
Fig. 4. Selectivity of this developed dopamine assay, DA concentration is 1 nM; BSA, HSA concentration is 1 mM; other substances are 1 μM (AA, ascorbic acid; CT, catechol; GL, glucose; Cys, L-cysteine; TR, tyrosine; PEA, phenethylamine; HSA, human serum albumin; BSA, bovine serum albumin; DA, dopamine; Mix, mixture of all interfering chemicals).
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